Single-Molecule RNA FISH in Whole-Mount Organoids

Area of Science:

• Cell Biology
• Molecular Biology
• Genomics

Background:

• Three-dimensional (3D) organoid cultures are advanced in vitro models that mimic tissue physiology.
• Single-molecule RNA fluorescent in situ hybridization (smFISH) is a powerful technique for detecting and quantifying individual RNA molecules.
• Integrating smFISH with organoid cultures presents technical challenges for visualizing RNA at the single-molecule level within complex 3D structures.

Purpose of the Study:

• To establish and validate a robust protocol for single-molecule RNA fluorescent in situ hybridization (smFISH) in 3D organoid cultures.
• To enable precise visualization and quantification of RNA expression within organoids.
• To provide a versatile method applicable to various biological investigations in organoid systems.

Main Methods:

• Developed a protocol for smFISH compatible with both whole-mount organoid staining and cryosectioning workflows.
• Utilized spinning disk confocal microscopy for whole-mount samples and widefield microscopy for cryosections.
• Optimized hybridization and washing steps for efficient RNA detection in 3D organoid structures.

Main Results:

• Successfully visualized and quantified single RNA molecules within diverse organoid models.
• Demonstrated the ability to identify specific cell types, such as intestinal stem cells using Lgr5 marker.
• Quantified RNA localization within the epithelial layer of organoids, revealing spatial expression patterns.

Conclusions:

• Organoid smFISH is an effective method for high-resolution RNA analysis in 3D culture models.
• The protocol supports diverse applications, including cell-type identification and spatial transcriptomics within organoids.
• This technique advances the study of gene expression and cellular heterogeneity in complex tissue models.